Note: We use Sigma Product: C2020 as our MALDI matrix, however we also recrystallize before use to purify it further. Sigma also sells a higher-grade MALDI matrix.
| Lab | Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
|---|---|---|---|---|
| Murphy & Metcalf | Formic Acid | Fisher Chemical | A117 | 99.5+%, Optima LC/MS Grade |
| Murphy & Metcalf | α-Cyano-4-hydroxycinnamic acid | Sigma | C2020 | ≥98% (TLC), powder |
| Murphy & Metcalf | Acetonitrile | Fisher | A9551 | LC-MS Ultra CHROMASOLV |
| Murphy & Metcalf | Methanol | Fisher Chemical | A456-4 | LC-MS Grade |
| Murphy & Metcalf | Water | Honeywell | LC365 | LC-MS Grade |
| Murphy & Metcalf | Trifluoroacetic acid | Fisher | A116 | LC-MS Grade |
| Metcalf | Toothpicks | NA | NA | Wood toothpicks are preferred |
| Murphy | Bruker Peptide Calibration standard | Fisher | NC9846988 | Bruker Daltonics 8206195 |
| Murphy | Bruker Daltonics Bacterial test standard | Fisher | NC0884024 | Bruker Daltonics 8604530. Note: This is not IVD BTS, which is for clinic-approved applications. |
| Murphy & Sanchez | 384-spot MALDI plates | Bruker Daltonics | varies | NA |
| Sanchez | Autoflex Speed LRF MALDI-TOF instrument | Bruker Daltonics | NA | Software is being continuously updated to support more vendor instruments |
Prepare the following solution and scale ingredients to the volume needed. Autoclave to sterilize.
If using 60 mm or larger petri-dishes, make a dilution-streak of a single strain on a single dish to to allow the formation of individual colonies. If using multi-well plates, plate each strain into 8 different wells.
Allow the bacteria to grow for a period of 7 days. (e.g. Inoculate agar plate on Monday -> put onto MALDI plate the following Monday).
If you haven’t already, download the Excel template here.
If you don’t have access to Microsoft Excel, you can use any other spreadsheet software such as: Apache OpenOffice™ “Calc”, which can be found at www.openoffice.org. When saving the file, ensure you save it as type “Microsoft Excel 97/2000/XP (.xls or .xlsx)”.
Before applying bacteria:
Sonicate or vortex the solution until no matrix crystals are observed. The matrix is then ready to be applied.
Use matrix solution within 1 week and store unused solid and prepared CHCA matrix between 2-8 °C, in the dark.
When applying bacteria to MALDI plate, your spot should resemble column 2. Column 1 has insufficient cell material, and column 3 has too much cell material
Each strain will have 8 biological replicates (8 MALDI spots). Each replicate is the result of touching a toothpick to a different colony/different part of a colony.
If using 60 mm or larger petri-dishes, create only one petri-dish per strain.
If using multi-well agar plates, create eight different wells per strain.
| 1. Apply bacteria directly without any prior chemical treatment. Smear a single bacterial colony in a thin layer directly onto the MALDI target plate using a sterile toothpick. | |
| 2. Leave the Calibration spots empty. | |
| 3. Add 1 µL of 70% formic acid to each spot containing bacteria. Let air dry. (~5 min) | |
| 4. Add 1 µL of MALDI matrix to each bacterial spot, let air dry. (~5 min) |
The MALDI plate should be properly cleaned before shipping. In order to clean the MALDI plate, use the steps below: method adapted from Freiwald & Sauer
Each plate will require multiple calibration spots. For protein data, follow the directions in the pdf link below: - Bruker Biotyper calibration procedure: - https://www.bruker.com/fileadmin/user_upload/8-PDF-Docs/Separations_MassSpectrometry/InstructionForUse/IFU_Bruker_Bacterial_Test_Standard_Revision_C.pdf
| Insert MALDI plate into the mass spectrometer |
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| Select the appropriate IDBac Method |
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| Under the “AutoXecute” control panel select “New”, which is to the right of “Run” |
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| If it wasn’t automatically detected, select the appropriate MALDI target plate geometry. |
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| Manually determine the minimum laser fluency/power needed and then press “Set initial laser power” before beginning the run |
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| Select “Calibrate with own template” and then select “New” |
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| Follow the directions in the left panel and then select “OK” |
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| Select “Next” |
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Within the “run parameters” page it is important to ensure the correct methods are selected in the correct places. For small molecule runs, change both autoXecute methods to: “IDBac_Small-Molecule_autoX”. For protein runs, change both autoXecute methods to: “IDBac_Protein_AutoX.axe”. There are four flexAnalysis methods: Either the protein or small molecule “…Calibrant Calibration” should be selected within the calibration box’s “flexAnalysis Method” pull-down menu. The matching protein or small molecule “…Unknown Sample Calibration” method should be selected within the second “flexAnalysis Method” pull-down menu. When you have finished, select “Next” |
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| Select “Save as” and save the sequence run to your data directory. Confirm and select “OK” |
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| Under the “AutoXecute” control panel select “Start automatic Run” |
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Dr. Brian T. Murphy
900 S. Ashland Ave. M/C 870
MBRB 3114
Chicago, IL 60607
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Return Address:
Dr. Brian T. Murphy
900 S. Ashland Ave. M/C 870
MBRB 3114
Chicago, IL 60607